Among the mesopelagic fishes, the lantern fishes (Family Myctophidae) are extremely common and numerous in both species and individuals in the open ocean midwaters of the world oceans. The myctophid fishes are generally found associated with the oxygen minimum layer of the midwaters and are active vertical migrators. They have adapted themselves to low oxygen conditions and low/dark light intensity conditions.
Most studies on midwater fish have been restricted to investigations of the oxygen minimum layer in the eastern tropical pacific and/or morphological adaptations (Kinzer et al. Deep Sea Research II, 40 (3): 783-800 (1993).
Little is known about the biology and ecology of this group particularly the dynamics of the interaction between mesozooplankton stocks and abundant migratory myctophids. The extreme oxygen deficiency of the Arabian Sea at mid depth has been well documented. Dissolved oxygen slowly increases below 1000 meter depth from about 0.2 ml per liter to 2.5 milliliter per liter at 2000 meter depth. Near the bottom at 3000 meter oxygen values average 3.5 ml per liter (SenGupta, R and S. W. A. Naqvi, Deep Sea Research, 31: 671-706, (1984).
The myctophid fish are adapted to low oxygen conditions and low/dark light intensity conditions. Their physiological adaptations to these varying abiotic and biotic factors have enabled them to survive in the vast sub-oxia below 150 meter depth zone.
The myctophids are strong vertical migrators and active swimmers. Most of them are black/dark brown in color and possess fluorescent organs called photophores. They constitute food for higher fishes of commercial importance such as Tuna, Sharks and marine mammals.
In the northern and western Arabian Sea itself the US GLOBEC Report No. 9 (1993) reported a biomass of about 100 million metric tonnes per year (present world fishery catch is around seventy million metric tonnes). Their dominance could be due to their ability to live in the suboxic middle depths and avoid predators.
Due to high protein content vast myctophid populations can form an excellent fish meal and poultry feed to support aquaculture and other farming activities. The myctophids can facilitate the assessment of water bodies, estimation of genetic resources, genetic variability and the level of gene flow between various stocks and populations in the world oceans.
Traditionally, various investigators including; Gunther, A. Report on the deep-Sea Fishes collected by H. M. S. Challenger during the years 1873-1876, Reprint 1963, Text volume, J. Cramer, Weinheim, Hafner, New York, 1-135 (1887); Gilbert, Proceedings of the U.S. National Museum, 48: 305-380 (1915), and Fraser-Brunner, proceedings of the Zoological society of London, 118 (4) 1019-1106 (1949), classified the species in the family Myctophidae by using differences in the position and numbers of photophores located on head and body of fish.
While photophore differences are still of primary importance, the morphometry is also used statistically (Paxton, Bulletin of the natural history museum of Los Angeles County, Science, No. 13 (1972).
The methods are incompetent to identify the closely allied species, populations and the life history stages. This is the major bottleneck for the proper identification, population dynamics and stock assessment of these fishes leaving their systematics unjustified.
There are several reasons for this confusion. In the first place, the fish are particularly fragile and easily damaged in collecting nets, so most specimens collected tend to be in poor condition.
Second, the group contains many morphologically similar species from all parts of the world.
Third though there is only small amount of variability in most characters, there is enough in some cases for similar species to overlap even in those characters wherein they differ most significantly (Zahuranec, B, Zoogeography and Systematics of the lantern fishes of the genus Nannobrachium (Lampanyctini: Myctophidae) PhD Thesis, Scripps Institution of Oceanography, University of California, San Diego, 1995).
The problems have been compounded since specimens of many forms have not been numerous. It is difficult to collect ample material for the taxonomic studies using the existing conventional methods.
The midwater habitat of the fish make them a uncommon and difficult material to get for work as special gears are required on board of ship.
The mesopelagic habitats of the north Arabian sea support rich and varied fauna. Most of these fishes belong to Myctophidae, Gonostomitids, Sternoptychids etc. The richness of this resource in the mesopelagic regime of the oceans all the world over has been well documented (Boltachev, A. R. J. Ichthyol, 27 (4): 539-547 (1987); Hussain and Ali Khan, Deep Sea Research, 34 (7A): 1293-1299 (1987); Alikhan and Aftab, Marine Research, 2 (1-2): 1-9 (1993).
US Globec, 1993 has reported a biomass of 100 million Tonnes of Myctophids in the Arabian Sea believed to be constituted by Benthosema pterotum and Diaphus sp. The true identity of stocks is, however, unknown.
In class Pisces there is a large body of population genetic data available on CD-ROMs and fish data bases (Agustin and Palomares, 5th International Symposium on Genetics in aquaculture, 19-25, 1994). However, very little information pertains to the families of lantern fishes. There is only one paper on myctophids by Afanas-yev et al. J. Ichthiology, 30 (1):28-37 (1990).
The earlier workers have described genetic diversity amongst conspecific and closely related species using protein electrophoresis. However, in the recent years, the molecular techniques using DNAs as the genetic markers have almost replaced these traditional methods with many shortcomings and providing information about only 1% of the genome.
Several workers have applied the new methods of DNAs for quantifying genetic relatedness among group of fish in stock assessments among wild and cultured populations and in studies of taxonomy and population genetic (Datta et al., Gene, 62: 331-336. 1988; Devlin et al. Canadian Journal of Fishery and Aquatic Sciences, 48 (9): 1606-1612,1991; Berson et al. Molecular Microbiology, 5 (9), 2261-2264, 1991; Martinez et al. Genome, 36: 1119-1123, 1993; Du Jun et al. DNA and cell Biology, 12 (8): 739-751, Cytogenetic and cellular genetics, 65: 233-237, 1993; Pogson et al. Genetics, 139: 375-3851994 and Carr et al. Molecular Ecology, 4:79-88, 1995.
There is a great lacunae in our knowledge of myctophid resource and stock assessment using genetic techniques. The only information so far available in related isozyme studies on 3 species endemic to the eastern pacific (Afanas-yev et al. 1990).
There is absolutely no literature available on genetic assessment of stocks from the other oceans.
A judicious exploitation and resource management requires proper identification of the larval and adult stages of huge myctophid populations in the world oceans. The genetic resource assessment, species identification, characterzation of life history stages, estimation of genetic variability and level of gene flow between various stocks for proper utiliztion of this vast fishery potential and for assessment and evaluation of trophic dynamics at the top end of the marine food chain.
Various authors have described methods of making DNA probes and their use as genetic markers in various organisms using mostly cDNA approach. Some patents pertain to improvement of DNA amplification related methods and formulation of different primers.
Weissman et al. 1983 patented Method for cloning genes (U.S. Pat. No. 4,394,443 published on Jul. 19, 1983). Mullis et al. In U.S. Pat. No. 4,683,195 published on Jul. 28, 1987 describe process for amplifying, detecting, and/or cloning nucleic acid sequences. The methods provided recombinant clones coding for human histocompatibility antigens, in particular clones for HLA-B antigens. The recombinant DNA expression system is developed for use in control of larval and adult insects and conferring pesticide resistance to crop plants.
Erlich et al. (U.S. Pat. No. 5,314,809 published on May 24, 1994) provide methods for enhanced specificity and sensitivity of nucleic acid amplification. The methods are simplified nested amplification procedures wherein both inner and outer primer pairs are present in the amplification reaction mixture.
Grosz, et al. In U.S. Pat. No. 5,340,728 published on Aug. 23, 1994 describe method for amplification of targeted segments of nucleic acid using nested polymerase chain reaction. Nuovo et al. (1996) in U.S. Pat. No. 5,538,871, published on Jul. 23, 1996 describe in improved In situ polymerase chain reaction. Barry et al. In U.S. Pat. No. 5,574,145 published on Nov. 12, 1996 isolated nucleic acid molecules targeted to the region intermediate to the 16 S and 23 S rRNA genes useful as probes for determining bacteria. They describe a method for generating DNA probes specific for an organism and capable of distinguishing in a non-empirical manner between species.
Cossart et al. (U.S. Pat. No. 5,523,205 published on Jun 4, 1996) describe DNA probes specific for hemolytic listeria bacteria. Trent et al. In the U.S. Pat. No. 5,693,464 published on Dec. 2, 1997 report rapid reproducible procedures for generating chromosome region-specific (CRSPs) for diagnostic and research applications.
Scott and Tomita (1998) give uses of cytochrome P450. Sub.Ipr gene (U.S. Pat. No. 5,734,086, published on Mar 31, 1998). Harris et al. (1998, U.S. Pat. No. 5,849,544, Dec. 15, 1998) give method of characterization and provide method for the detection of diagnostic base sequences in one or more nucleic acids contained in a sample.
Jeffreys et al (1998, U.S. Pat. No. 5,853,989, published on, Dec 29, 1998) describe method of characterisation of genomic DNA. They used primers which selectively prime specific type of internal repeat unit in a tandemly repeated region. Ryder et al. (1998, U.S. Pat. No. 5,786,183, published on Jul 28, 1998) give methods of enhancing nucleic acid amplification. Dandliker et al. (1998, U.S. Pat. No. 5,707,813, published on Jan 13, 1998) report nucleic acid probes and methods. Kuhns (1999, U.S. Pat. No. 5,981,171, published on Nov. 9, 1999) describe diagnostic assays using nucleic acid probes. He describes methods and compositions for a rapid quantitative nucleic acid hybridization assay fpr detecting a DNA or RNA sequence in a biological sample.
Caetano-Anolles (1999, U.S. Pat. No. 5,962,221, published on Oct. 5, 1999) give oligonucleotide constructs and methods for the generation of sequence signatures from nucleic acids.
Bebbington et al (1999, U.S. Pat. No. 5,891,693, published on Apr. 6, 1999) describe recombinant DNA methods vectors and host cells. Rothschild et al. In U.S. Pat. No. 5,939,264 published on Aug 17, 1999) describe genetic markers in pigs for reproductive traits using polymorphism in the reproductive genes.
Molecular cloning and characterization of a further gene sequences coding for human relaxin is given in U.S. Pat. No. 4,758,516 by Hudson et al. published on Jul. 19, 1988.
Some patents related to the genes reported by us is available on humans and other organisms. Aguirre et al. (1998) developed progressive rod-cone degeneration disease genetic markers and assays in a canine (U.S. Pat. No. 5,804,388). Reports on diagnosis of hereditary retinal degenerative diseases is given by Dryja et al. In U.S. Pat. No. 5,262,529 published on Nov. 16, 1993 They developed a probe for identifying region of photoreceptor protein of humans and also made the primers. Dryja et al in Mar. 1996, U.S. Pat. No. 5,498,521 published on Mar. 12, 1996 later report the method which involves analyzing the DNA of the subject to determine the presence or absence of a mutation in a gene for photoreceptor protein. Shassere, et al. (1997 U.S. Pat. No. 5,698,398 published on Dec. 16, 1997) disclosed quality control compositions suitable as sample specimens to measure performance of DNA probe tests which determine cytogenetic abnormalities, such as chromosome copy number, of cells in a tissue sample.
However, all of these are related to making cDNA probes and the work is mostly on humans, canines, bacteria and other organisms, mostly of terrestrial origin.
No patent is available on fish DNA probes and sequences as genetic markers particularly in the myctophid fish. Nor are primer sequences designed for these fishes.
Earlier Mitochondrial DNA control region and Ribosomal Internal transcribed spacer (ITS2) are used for systematic relationships. Reed K. M, et al. (1998) studied sequence analysis of the mitochondrial DNA control region of ciscoes (genus Coregonus) with taxonomic implications for the Great Lakes species flock.
Molecular cloning of rod opsin (rhodopsin) cDNA from retinas of various teleost fishes, octopus, squids, shrimps, Lamprey and screening of cDNA libraries of rhodopsin is done by several workers (O'Brien and Al-Ubaidi, M. R., Gene, 193 (2): 141-150; Crescitclli, F, et al. Journal of Comparative Physiology, 1985: 157 (3): 323-333; Tsai, H. J. et al., Biochemical molecular Biology, 109, 91: 81-88; Hara-Nishimura et al. FEBS-LETT, 317 (1-2): 5-11, 1993). Crandall, K. A. and Hillis, D. M. Nature, 387 (No. 6634), 667-668,1997, describe rhodopsin protein evolution in the dark in cave dwelling cray fish. Fitzgibbon, j et al., Gene, 1995,164 (2), 273-277. Harada, Y et al. Journal of Biochemistry, 110 (4), 501-507,1991 report synthesis and expression of rhodopsin gene in Octopus. But all these reports deal with the protein rhodopsin, not much has been said about its gene even in these species.
Douzery E, et al. Molecular Biology and Evolution, 14(11):1154-66 (1997) use the mitochondrial control region of Cervidae: evolutionary patterns and phylogenetic content. Barreto G, et al. American Journal of Human Genetics 58(3):609-16 (1996) report Mitochondrial D-loop “signatures” produced by low-stringency single specific primer PCR constitute a simple comparative human identity test. Brown JR, et al. Genetics. 142(2):525-35. 1996 describe Length variation, heteroplasmy and sequence divergence in the mitochondrial DNA of four species of sturgeon (Acipenser). Lee W J, et al. J Mol Evol. July 1995;41(1):54-66 report structure and evolution of teleost mitochondrial control regions. No reports of DNA probes for genetic marking of any marine species is come across.
Jobst J, et al (1998) give information on Molecular evolution of the internal transcribed spacers (ITS1 and ITS2) and phylogenetic relationships among species of the family Cucurbitaceae. Odorico D M, et al. (1997) describe Variation in the ribosomal internal transcribed spacers and 5.8S rDNA among five species of Acropora (Cnidaria; Scleractinia): patterns of variation consistent with reticulate evolution. Despres L, et al (1995) describe ITS2 ribosomal RNA indicates Schistosoma hippopotami is a distinct species. Crabtree M B, et al. (1995) study development of a species-diagnostic polymerase chain reaction assay for the identification of Culex vectors of St. Louis encephalitis virus based on interspecies sequence variation in ribosomal DNA spacers. Mukhamedov R S, et al. (1994) report [Nucleotide sequence of internal transcribed spacers and 5.8S rDNA for the ribosomal operon from alfalfa Medicago sativa and cotton Gossypium hirsutum L]. No reports of DNA probes in open ocean marine fish are seen.
Goswami and Bernardi (1999) described nucleotide gene sequences of 16 S and 12 S genes of five myctophid species viz: Tarletonbeania crenularis; Protomyctophum crockeri; Lampanyctus regalis; Diaphus theta; Stenobrachius leucosparus in the National center of Biotechnology information GenBank. These sequences were published on 20 Apr., 1999 and can be screened at www period ncbi period nlm period nih period gov Genbank databases. The respective accession numbers are AF134250; AF134249; AF134248; AF134247; AF134246; AF134245; AF134244; AF134243; AF134242 and AF134241.
As such, in order to obcriate the draw backs listed in the use of conventional taxonomic methods for identification of open ocean midwater fish, the applicants developed a novel method and approach, using molecular biology.
The invention relates to the molecular cloning and characterization of specific gene region sequences. More particularly the invention relates to genetic markers which have been identified in several genes such as Cytochrome b (cyt b); Mitochondrial control region (D-Loop); Internal Transcribed spacer between nuclear ribosomal genes (ITS2) & Rhodopsin gene of visual pigments (Rod) and mitochondrial ribosomal RNA genes for myctophid fishes.
As discussed above, prior art patents and reports deal with different groups of organisms. The invention on the other hand concerns an economically important group of open ocean midwater fish found in all the world oceans which has adapted itself to the oxygen minimum conditions and to low/dark light intensities for vision, the genetic information of which has not been reported so far.
The genomic DNA probes are sequence specific and are ideal for identification of complimentary regions of species specific genes. Cytochrome b is an important component of mitochondrial electron transport chain and plays important role in respiratory physiology (William, F. Ganong, Chapter 17 199-235 in Review of medical physiology pp. 599; 1977). The gene encoding cytochrome b, termed Cyt b of myctophid fishes plays an important role in adaptations and survivals of these fishes in the suboxic regions.
The Rod gene encoding for rhodopsin of the retinal rods is important for adapations of these fishes to varying light intensities which varies with the depth.
The mitochondrial control region D-Loop an Internal transcribed spacer ITS2 gene regions are highly prone to rapid mutations and can be suitable gene candidates for identification of variants at the intra species and population level.
The invention discloses species specific DNA probes for detection of genes such as cyt b, Rod, D-Loop, ITS2. This invention is useful for identification of early and adult life history stages of myctophids i.e. Lantern fishes.
This is the first report of preparation of DNA probes useful for molecular genetic identification of myctophid fish. Novelty of the invention is in use of novel gene region DNA probes for identification of eggs and early larvae of mesopelagic fish which live in oxygen minimal and low light conditions.